This invention relates to neuroprobes for mapping monoamine reuptake sites in the brain, and particularly to neuroprobes that can also serve as radiotracers for use in single-photon emission computed tomography (SPECT) and positron emission tomography (PET) for imaging of such reuptake sites.
A brain consists of a plurality of neurons that interact by exchanging chemical messengers. Each neuron generates neuro-chemicals, referred to as neurotransmitters; neurotransmitters act at sites on the cellular membrane of a neuron, the sites being referred to as receptors. Receptors are associated with either ion channels through the cellular membrane or secondary neurochemical messenger systems. By contrast, reuptake sites are molecular complexes which transport chemicals across the cellular membrane of a neuron. When a neurotransmitter has served its function, it is removed from the vicinity of the receptor by being bound to a reuptake site which transports the neurotransmitter to the interior of the neuron.
Just as there are many specialized neurons in the brain, there are also a variety of neurotransmitters, associated receptors, and reuptake sites. The distribution of specialized neurons depends upon the particular organism under study, and the state of health of that organism.
A neuron can be classified according to the type of neurotransmitter that it uses to communicate with other neurons. Certain types of neurons can be found predominantly in particular regions of the brain. For example, the striatal region of a mammalian brain is innervated by neurons using dopamine as a neurotransmitter. The striatum also contains a large number of non-dopaminergic neurons that have dopamine receptors. Certain compounds, such as cocaine, have a preferential affinity for dopamine reuptake sites, and therefore tend to bind to such reuptake sites. The effect of a molecule such as cocaine upon a dopamine reuptake site is to inhibit reuptake of the neurotransmitter dopamine, leaving more dopamine available in the vicinity of the dopamine receptors.
In certain neurological diseases, such as Parkinson""s disease, distinct groups of neurons lose their normal physiological functioning. Consequently, the abnormal neurons may behave differently in the presence of some neurotransmitters, and may also produce neurotransmitters in a manner that differs from a healthy neuron.
The major neurotransmitters, dopamine, norepinephrine, and serotonin, are referred to collectively as the monoamine neurotransmitters. Many neurons have receptors adapted to receive at least one of these neurotransmitters. Parkinson""s disease is caused by the degeneration of some of the dopaminergic neurons in the brain. The neurons lost in Parkinson""s disease have a large number of dopamine reuptake sites; cocaine and chemical analogs of cocaine have an affinity for such reuptake sites.
A radioisotope is commonly incorporated in molecules that have a demonstrated binding affinity for a particular type of neuro-receptor, and such molecules are commonly used as neuroprobes. The localization of neuroprobes can be used to find specialized neurons within particular regions of the brain. It is also known that a neurological disease can be detected by observing abnormal binding distributions of a neuroprobe. Such abnormal binding distributions can be observed by incorporating a radionuclide within each molecule of the neuroprobe with a high binding affinity for the particular reuptake sites of interest. Then, an imaging technique can be used to obtain a representation of the in vivo spatial distribution of the reuptake sites of interest.
In single photon emission computed tomography (SPECT) imaging, the most commonly used radionuclides are heavy metals, such as 99mTc. Heavy metals are very difficult to incorporate into the molecular structure of neuroprobes because such probes are relatively small molecules (molecular weight less than 400).
In positron emission tomography (PET), the radiohalide 18F (fluorine) is commonly used as a substitute for H (hydrogen) in radiopharmaceuticals because it is similar in size. Not all halogens will work, however. For example, I (iodine) is much larger than both H and F, being approximately half the size of a benzene ring. However, due to the small size of typical radiopharmaceuticals for use as neuroprobes, the presence of iodine markedly changes the size of the compound, thereby altering or destroying its biological activity.
In addition, the presence of iodine in a neuroprobe tends to increase its lipophilicity, and therefore increases the tendency of the neuroprobe to engage in non-specific binding. For example, paroxetine is a drug with high affinity and selectivity forserotonin reuptake sites, and [3H]paroxetine has been shown in rodents to be a useful in vivo label (Scheffel, U. and Hartig, P R. J. Neurochem., 52: 1605-1612, 1989). However, several iodinated analogs of this compound with iodine attached at several different positions had unacceptably low affinity, in fact being one tenth of the affinity of the parent compound. Furthermore, when the iodinated compound was used as an in vivo radiolabeled neuroprobe, non-specific binding activity was found to be so high that no measurable portion of the brain uptake appeared to be specifically bound to the serotonin reuptake site. Thus, the iodinated form of paroxetine is not useful as an in vivo probe.
The addition of iodine to a neuroprobe can unfavorably alter its biological properties. For example, tomoxetine has high affinity and selectivity for norepinephrine reuptake sites. However, when tomoxetine is iodinated, e.g. to form R-4-iodoto-moxetine, the resulting labeled compound has low affinity for such reuptake sites, and relatively high affinity for serotonin reuptake sites. In vivo labeling studies have shown that it is an unacceptably poor probe even for the serotonin reuptake sites because it exhibits low total brain uptake and immeasurably low specific uptake.
An iodinated compound can be useful as an in vitro probe, but may be useless as an in vivo probe, because an in vivo probe must meet the requirements associated with intravenous administration of the probe to a living subject. Reasons for the loss of in vivo utility include the fact that the compound may be metabolized too quickly, that it may not cross the blood-brain-barrier, and that it may have high non-specific uptake into the lipid stores of the brain. In vitro homogenate binding studies remove these obstacles by isolating the brain tissue from hepatic metabolic enzymes, by homogenizing the brain tissue so as to destroy the blood-brain-barrier, and by diluting the brain tissue so as to decrease the concentration of lipids in the assay tube. Accordingly, it cannot be assumed that a probe will be useful in both in vivo and in vitro modalities.
An in vivo SPECT probe was developed by iodinating cocaine. However, this probe shows a binding affinity and specificity no better than cocaine itself, which is inadequate for purposes of SPECT imaging.
In one aspect, the invention is directed to a neuroprobe for mapping monoamine reuptake sites, having the formula: 
wherein R can be aryl, substituted aryl, heterocyclic, CO(CH2)nY, (CH2)CHF2, and (CH2)nY. Y can be Cl, Br, I, (CH2)m, aryl, substituted aryl, heterocyclic, CO2H, CO2R3, CO2NR3R4, OH, OR3, CH(OR3)2, CR3(OR4)2, OCOR3, OSO2R3, OCONR3R4, OCOOR3, CONR3R4, NR3R4, NR3COR4, NR3CO2R4, NR3CONR4R5, NCS, NCO. R3, R4 and R5 can be alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, or heterocyclic; m=3-8 and n=1-6. Rxe2x80x2 can be CwH2w+1 wherein w=0-6 and C includes an isotope of carbon, including at least one radioactive isotope of carbon. CO2Rxe2x80x2 can be in the xcex2 position as shown or, further, CO2Rxe2x80x2 can be in the a position. X can be an isotope of Cl, an isotope of Br, an isotope of F, an isotope of I, or Sn(Rxe2x80x31Rxe2x80x32Rxe2x80x33), wherein Rxe2x80x31, Rxe2x80x32, and Rxe2x80x33 are CpH2p+1 groups where p=1-6, or an aryl group.
In a second aspect, the invention is directed to an iodinated neuroprobe for mapping monoamine reuptake sites, having the formula: 
wherein R can be a CnH2n+1 group where n=0-6, an alkenyl group, or a mCnH2n+1 group where n=1-6 and where m=11 or 14 for at least one mC. Rxe2x80x2 can be a CwH2w+1 group where w=0-6, a p-iodophenylmethyl group, a p-iodophenylethyl group, a phenylmethyl group, or a phenylethyl group. CO2Rxe2x80x2 can be in the xcex2 position as shown or, further, CO2Rxe2x80x2 can be in the xcex1 position. X can be an isotope of F, an isotope of Cl, an isotope of Br, an isotope of I, CH3, or Sn(Rxe2x80x31Rxe2x80x32Rxe2x80x33). Rxe2x80x31 can be a CpH2p+1 group where p=1-6, or an aryl group; Rxe2x80x32 can be a CpH2p+1 group where p=1-6, or an aryl group; and Rxe2x80x33 can be a CpH2p+1 group where p=1-6, or an aryl group. Y can be H only if X is an isotope of I, or Rxe2x80x2 is a p-iodophenylmethyl group, or Rxe2x80x2 is a p-iodophenylethyl group, else Y=an isotope of I.
In a third aspect, an iodinated neuroprobe of the invention has the formula: 
wherein R can be a CnH2n+1 group where n=0-6, an alkenyl group, or a mCnH2n+1 group where n=1-6 and where m=11 or 14 for at least one mC. Rxe2x80x2 can be a CwH2w+1 group where w=0-6, a p-iodophenylmethyl group, a p-iodophenylethyl group, a phenylmethyl group, or a phenylethyl group. CO2Rxe2x80x2 can be in the xcex2 position as shown or, further, CO2Rxe2x80x2 can be in the xcex1 position. X can be an isotope of F, an isotope of Cl, an isotope of Br, an isotope of I, CH3, or Sn(Rxe2x80x31Rxe2x80x32Rxe2x80x33). Rxe2x80x31 can be a CpH2p+1 group where p=1-6, or an aryl group; Rxe2x80x32 can be a CpH2p+1 group where p=1-6, or an aryl group; and Rxe2x80x33 can be a CpH2p+1 group where p=1-6, or an aryl group. Y can be H only if X is an isotope of I, or Rxe2x80x2 is a p-iodophenylmethyl group, or Rxe2x80x2 is a p-iodophenylethyl group, else Y=an isotope of I. W can be O, S, (CH2)q, O(CH2)q where q=1-6, wherein X resides on a benzene ring of the formula at an ortho, meta, or para position with respect to W, and Y resides at any remaining position on the benzene ring.
For each of the foregoing embodiments there is provided a precursor of the radiolabeled neuroprobe that lacks a radiotracer atom, and a kit for preparing an associated iodinated neuroprobe. Also included are derivatives of the neuroprobes that include 18F substituted onto R.
Intermediates useful for the preparation of the iodinated neuroprobes of the second and third aspects of the invention advantageously have the same formula as the final products, only R=H.
As used herein xe2x80x9cxcex2-CITxe2x80x9d and xe2x80x9cCITxe2x80x9d refer to 2xcex2-carbomethoxy-3xcex2-(4-iodophenyl)tropane. As used herein, the substituent (CH2)m refers to a cycloalkyl group, e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. As used herein, the abbreviation (DAT) refers to the dopamine transporter; the abbreviation NET refers to the norepinephrine transporter; 5-HTT refers to 5-hydroxytryptamine, or 5-HT, transporter.
Both the radiostable and radioactive variants of the iodinated neuroprobe of the invention are useful for human and non-human research. For example, in vivo and in vitro experiments can be performed using the compounds of the invention to study monoamine reuptake sites generally, and cocaine binding sites in particular.